依诺肝素钠骨水泥通过诱导 M2 巨噬细胞极化发挥抗炎免疫调节作用。

OBJECTIVE: The implantation of PMMA bone cement results in an immune response and the release of PMMA bone cement particles causes an inflammatory cascade. Our study discovered that ES-PMMA bone cement can induce M2 polarization of macrophages, which has an anti-inflammatory immunomodulatory effect. We also delved into the molecular mechanisms that underlie this process. METHODS: In this study, we designed and prepared samples of bone cement. These included PMMA bone cement samples and ES-PMMA bone cement samples, which were implanted into the back muscles of rats. At 3, 7, and 14 days after the operation, we removed the bone cement and a small amount of surrounding tissue. We then performed immunohistochemistry and immunofluorescence to observe the polarization of macrophages and the expression of related inflammatory factors in the surrounding tissues. The RAW264.7 cells were exposed to lipopolysaccharide (LPS) for 24 h to establish the macrophage inflammation model. Then, each group was treated with enoxaparin sodium medium, PMMA bone cement extract medium, and ES-PMMA bone cement extract medium, respectively, and cultured for another 24 h. We collected cells from each group and used flow cytometry to detect the expressions of CD86 and CD206 in macrophages. Additionally, we performed RT-qPCR to determine the mRNA levels of three markers of M1 macrophages (TNF-α, IL-6, iNOS) and two M2 macrophage markers (Arg-1, IL-10). Furthermore, we analyzed the expression of TLR4, p-NF-κB p65, and NF-κB p65 through Western blotting. RESULTS: The immunofluorescence results indicate that the ES-PMMA group exhibited an upregulation of CD206, an M2 marker, and a downregulation of CD86, an M1 marker, in comparison to the PMMA group. Additionally, the immunohistochemistry results revealed that the levels of IL-6 and TNF-α expression were lower in the ES-PMMA group than in the PMMA group, while the expression level of IL-10 was higher in the ES-PMMA group. Flow cytometry and RT-qPCR analyses revealed that the expression of M1-type macrophage marker CD86 was significantly elevated in the LPS group compared to the NC group. Additionally, M1-type macrophage-related cytokines TNF-α, IL-6, and iNOS were also found to be increased. However, in the LPS + ES group, the expression levels of CD86, TNF-α, IL-6, and iNOS were decreased, while the expression of M2-type macrophage markers CD206 and M2-type macrophage-related cytokines (IL-10, Arg-1) were increased compared to the LPS group. In comparison to the LPS + PMMA group, the LPS + ES-PMMA group demonstrated a down-regulation of CD86, TNF-α, IL-6, and iNOS expression levels, while increasing the expression levels of CD206, IL-10, and Arg-1. Western blotting results revealed a significant decrease in TLR4/GAPDH and p-NF-κB p65/NF-κB p65 in the LPS + ES group when compared to the LPS group. Additionally, the LPS + ES-PMMA group exhibited a decrease in TLR4/GAPDH and p-NF-κB p65/NF-κB p65 levels when compared to the LPS + PMMA group. CONCLUSION: ES-PMMA bone cement is more effective than PMMA bone cement in down-regulating the expression of the TLR4/NF-κB signaling pathway. Additionally, it induces macrophages to polarize towards the M2 phenotype, making it a crucial player in anti-inflammatory immune regulation.

目的：PMMA 骨水泥的植入会引发免疫反应，PMMA 骨水泥颗粒的释放会引发炎症级联反应。我们的研究发现，ES-PMMA 骨水泥可以诱导巨噬细胞向 M2 极化，从而产生抗炎免疫调节作用。我们还探讨了这一过程的分子机制。

方法：在这项研究中，我们设计并制备了骨水泥样本。包括 PMMA 骨水泥样本和 ES-PMMA 骨水泥样本，将其植入大鼠的背部肌肉中。术后 3、7 和 14 天，取出骨水泥和少量周围组织。然后进行免疫组织化学和免疫荧光染色，观察周围组织中巨噬细胞的极化和相关炎症因子的表达。用脂多糖（LPS）处理 RAW264.7 细胞 24 小时，建立巨噬细胞炎症模型。然后，将各组分别用依诺肝素钠培养基、PMMA 骨水泥浸提液培养基和 ES-PMMA 骨水泥浸提液培养基处理，再培养 24 小时。收集各组细胞，用流式细胞术检测巨噬细胞中 CD86 和 CD206 的表达。此外，还进行了 RT-qPCR 检测 M1 巨噬细胞（TNF-α、IL-6、iNOS）和 M2 巨噬细胞（Arg-1、IL-10）的三个标志物的 mRNA 水平。进一步通过 Western blot 分析 TLR4、p-NF-κB p65 和 NF-κB p65 的表达。

结果：免疫荧光结果表明，与 PMMA 组相比，ES-PMMA 组的 M2 标志物 CD206 上调，M1 标志物 CD86 下调。此外，免疫组织化学结果显示，ES-PMMA 组的 IL-6 和 TNF-α 表达水平低于 PMMA 组，而 ES-PMMA 组的 IL-10 表达水平较高。流式细胞术和 RT-qPCR 分析表明，与 NC 组相比，LPS 组的 M1 型巨噬细胞标志物 CD86 表达显著升高。此外，M1 型巨噬细胞相关细胞因子 TNF-α、IL-6 和 iNOS 也增加。然而，在 LPS+ES 组中，CD86、TNF-α、IL-6 和 iNOS 的表达水平降低，而 M2 型巨噬细胞标志物 CD206 和 M2 型巨噬细胞相关细胞因子（IL-10、Arg-1）的表达水平增加。与 LPS+PMMA 组相比，LPS+ES-PMMA 组的 CD86、TNF-α、IL-6 和 iNOS 表达水平下调，而 CD206、IL-10 和 Arg-1 的表达水平上调。Western blot 结果显示，与 LPS 组相比，LPS+ES 组 TLR4/GAPDH 和 p-NF-κB p65/NF-κB p65 显著降低。此外，与 LPS+PMMA 组相比，LPS+ES-PMMA 组 TLR4/GAPDH 和 p-NF-κB p65/NF-κB p65 水平降低。

结论：ES-PMMA 骨水泥比 PMMA 骨水泥更能有效下调 TLR4/NF-κB 信号通路的表达。此外，它诱导巨噬细胞向 M2 表型极化，在抗炎免疫调节中发挥重要作用。